Flight of a Cytidine Deaminase Complex with an Imperfect Transition State Analogue Inhibitor: Mass Spectrometric Evidence for the Presence of a Trapped Water Molecule

Cytidine deaminase (CDA) binds the inhibitor zebularine as its 3,4-hydrate (K_d ∼ 10^–12 M), capturing all but ∼5.6 kcal/mol of the free energy of binding expected of an ideal transition state analogue (K_tx ∼ 10^–16 M). On the basis of its entropic origin, that shortfall was tentatively ascribed to the trapping of a water molecule in the enzyme–inhibitor complex, as had been observed earlier for product uridine [Snider, M. J., and Wolfenden, R. (2001) Biochemistry 40, 11364–11371]. Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) of CDA nebularized in the presence of saturating 5-fluorozebularine reveals peaks corresponding to the masses of E₂Zn₂W₂ (dimeric Zn-CDA with two water molecules), E₂Zn₂W₂Fz, and E₂Zn₂W₂Fz₂, where Fz represents the 3,4-hydrate of 5-fluorozebularine. In the absence of an inhibitor, E₂Zn₂ is the only dimeric species detected, with no additional water molecules. Experiments conducted in H₂¹⁸O indicate that the added mass W represents a trapped water molecule rather than an isobaric ammonium ion. This appears to represent the first identification of an enzyme-bound water molecule at a subunit interface (active site) using FTICR-MS. The presence of a 5-fluoro group appears to retard the decomposition of the inhibitory complex kinetically in the vapor phase, as no additional dimeric complexes (other than E₂Zn₂) are observed when zebularine is used in place of 5-fluorozebularine. Substrate competition assays show that in solution zebularine is released from CDA (k_off > 0.14 s^–1) much more rapidly than is 5-fluorozebularine (k_off = 0.014 s^–1), despite the greater thermodynamic stability of the zebularine complex.